Background and Objectives. Malaria infection, caused by Plasmodium falciparum, is the most lethal and frequently culminates in severe clinical complications. Interleukin-22 (IL-22) has been implicated in several diseases including malaria. The objective of this study was to investigate the role of IL-22 gene polymorphisms in P. falciparum infection. Material and Methods. Ten single-nucleotide polymorphisms (SNPs), rs976748, rs1179246, rs2046068, rs1182844, rs2227508, rs2227513, rs2227478, rs2227481, rs2227491, and rs2227483, of IL-22 gene were genotyped through PCR-based assays of 250 P. falciparum infection. IL-22 gene promoter activity. P. P. falciparum infection. P. P. P. P.  We found that the rs2227481 TT genotype (odds ratio 0.254, confidence interval = 0.097-0.663, P. falciparum infection. IL-22 gene promoter activity.  The study suggests that IL-22 polymorphisms in rs2227481 and rs2227483 could contribute to protection against  Plasmodium falciparum is associated with significant morbidity and mortality in humans [Malaria caused by n humans . It affen humans . Malarian humans .Eliminating Countries for 2020 (E-2020)” initiative of the WHO to achieve the goal of a malaria-free country by 2020 [Plasmodium species causing the majority of the malaria cases is P. falciparum (97%) followed by P. vivax (2%).Saudi Arabia is one of the largest countries in the Middle East with a population of over 32 million. The implementation of malaria control measures in Saudi Arabia over the past 50 years, in collaboration with the World Health Organization (WHO), has effectively reduced the magnitude of risk throughout the nation –6. In 20P. falciparum infection. Also known is the fact that malaria parasites have a robust selective pressure on human genetics, particularly in regions where malaria is endemic [P. falciparumare influenced by host genetic factors and assist in establishing the pathological mechanisms underlying the vulnerability to the infection [α) gene are associated with an increase in SMA in P. falciparum-infected patients [α gene have been shown to increase the susceptibility to severe malaria [It is known that the host genetic polymorphisms play an important role in the variation of malaria severity associated with  endemic , 8. Varinfection . For exapatients . SNPs in malaria .IL-22 gene is positioned at chromosome 12q15. Both innate and adaptive arms of the immune system produce IL-22, including αβ T, γδ T, NKT, and innate lymphoid cells (ILCs). Certain nonhematopoietic and myeloid cells can also release IL-22 [Interleukin-22 (IL-22) has been classified as a cytokine of the IL-20 subfamily. The se IL-22 , psoriasse IL-22 , inflammse IL-22 , Guillaise IL-22 .IL-22 gene are associated with several diseases such as ulcerative colitis (UC) [P. chabaudi malaria infection in mice, a higher production of IL-22 was observed in the liver, which is important as it suggests that this cytokine is an essential factor in the protection against the lethal infection caused by P. chabaudi [P. falciparum-associated malaria and pathogenesis. Therefore, this report investigates the plausible role of variations at the IL-22 gene in the complications associated with P. falciparum infection in a Saudi Arabian population.Polymorphisms in the tis (UC) , psoriastis (UC) , bladdertis (UC) , and systis (UC) . It has tis (UC)  and an Ichabaudi . Such a P. falciparum-infected patients admitted to the Malaria Center in the Jazan region, located in the southwest of Saudi Arabia. The study also included 200 randomly selected uninfected healthy individuals as a control group. A thick blood film from each infected patient was used to determine the parasite density according to the “plus system” scale, described in the WHO manual [Venous blood was collected from 250 O manual . Based oThis study was approved by the Research Ethics Committee at King Fahad Hospital (KFCH), Jazan (Registry number 041) and conducted in accordance with the Helsinki Declaration of 1975. Informed consent were obtained from all participants prior to their participation in the study; consent for children were given by their legal guardians. To ensure confidentiality, patient data and biological samples were anonymized prior to processing by laboratory staff. Basic demographic and clinical data required to accomplish the objectives of this study were collected and securely retained for all study participants.IL-22 gene region were selected for genotyping. The IL-22 SNPs included were rs976748, rs1179246, rs2046068, rs1182844, rs2227508, rs2227513, rs2227478, rs2227481, rs2227491, and rs2227483. Candidate SNPs were selected based on the following criteria: (i) a minor allele frequency (MAF) ≥ 5%, as reported by the 1000 genome for the combined populations and (ii) a linkage disequilibrium (LD) threshold (r2) ≤ 0.8 or (iii) existence of evidence for an association with malaria. Marker information of the SNPs selected is shown in Supplementary A total of ten SNPs within the http://bioinfo.ut.ee/primer3-0.4.0/). All primers were tailed with an M13 sequence to facilitate the sequencing of the PCR products. Sequences of the primers and PCR conditions used for each SNP are shown in Supplementary DNA was extracted using the DNeasy Blood and Tissue kit  according to the manufacturer's protocol and stored at -20°C for further experimentation. The PCR was performed using the GoTaq Green Master Mix PCR . Primers flanking SNPs of interest were designed using Primer3 Input (version 0.4.0)  following the manufacturer's instructions. Extracted plasmids with cloned fragments were transfected into HEK293 cells (Human Embryonic Kidney 293 cells), which were harvested and tested for luciferase activity after 48 hours.Gene fragments of the promoter region containing IL-22 rs2227513 A/G variants were amplified and cloned in the pGL3 Basic Vector . Primers were tailed at the 5′ end with specifying endonuclease recognition sites for the restriction enzymes SmaI and BglII . In addition, a few overhang sequences (CGCCTA) were added to facilitate restriction enzyme binding. Primers used for the amplification of the promoter region, PCR conditions, and chromosomal locations are shown in Supplementary http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) was used to conduct a genotypic association test for all the selected variants as the primary test of association. We also performed the allelic, dominant, and recessive model for each variant to explore the possibility of other models of inheritance. The results were expressed as odds ratios (OR) with 95% confidence intervals (95% CI). Testing of deviation from the Hardy-Weinberg equilibrium (HWE) was performed; rs2227513 was excluded from further analyses due to the significant deviation from HWE . The test of association was considered significant if the P value, obtained in a two-tailed test, was <0.05.The SNPs were analyzed and measures of LD, haplotype frequencies, and MAFs were calculated using the Haploview software (version 4.0) . The de Finetti program  . We also found that the T allele was associated with a lower risk of P. falciparum malaria in a recessive model . For IL-22 variant rs2227483, we identified that the heterozygous AT genotype frequency was significantly higher in the control group in comparison to the patient group .The genotype distribution of the SNPs in the IL-22 gene between the = 0.002) . In addiχ2 = 6.13, and P = 0.013) . The IL-22 SNPs were examined in cohort I compared to cohort II+III+IV and cohort I+II compared to cohorts III+IV; however, none of the SNPs showed any statistically significant distribution between the groups  . In addiχ2 = 4.14, and P = 0.044). Also, the rs2046068 A allele was significant in a dominant model   .χ2 = 5.14, and P = 0.023) . The rs2227478 CT genotype was substantially elevated in the 10 years and older age group malaria compared to the younger age group  , with the T allele of rs2227483 associated with the 10 years and older age group .Comparing the malaria group in a 1-9 years age group with a 10 years and older age group, the frequency of the rs976748 CT genotype was significantly higher in the 1-9 years age group compared to the other group  . Under a= 0.030) . SimilarP. falciparum malaria comparing the malaria group in the 10-20 years age group with the 21 years and older group . No dominant or recessive statistically significant relationship was observed between the two groups. In contrast, the IL-22 rs2227483 T allele was associated with a high susceptibility to develop P. falciparum malaria in the 41 years and older age group with a higher frequency of the rs2227483 T allele . For both SNPs, no dominant or recessive relationship was detected.No relationship was observed between the IL-22 SNPs and the risk of ge group . We founP. falciparum-infected group and the control group and two blocks were generated. Block 1 comprised the IL-22 polymorphisms rs1179246, rs1182844, and rs976748, with block 2 consisting of IL-22 polymorphisms rs2046068 and rs2227491 (P = 0.034). In block 2, one haplotype A-G of rs2046068 and rs2227491 was statistically significant and prevalent in the control group but not in the malaria group .A haplotype analysis was done between the s2227491 . One hapP = 0.036).A haplotype analysis was performed between cohort I+II and cohort III+IV of varied parasite density . The disP = 0.056). However, the haplotype C-A of rs2046068 and rs2227491 was significantly high in the 6 years and older age group .A haplotype distribution analysis was done in the malaria group for two age groups, 1-5 years and 6 years and older. The frequency of two haplotypes was found to be either statistically significant or close to significance . The hapP = 0.041).A haplotype analysis was also performed between patients in the 1-9 years and 10 years and older age groups. One single haplotype was statistically significant . The hapP = 0.045).Finally, we assessed the haplotype distribution between patients in the 1-9 years and 41 years and older age groups. The distribution of two haplotypes was significantly different in these two groups . The hapFor the delineation of the molecular mechanisms driving the IL-22 gene, nested deletions from the IL-22 promoter were generated  and clonP < 0.0001).To test whether rs2227513 contributes to the expression of the IL-22, one nucleotide was changed in clone F3/R1 . The IL-P. falciparum and has an efficacy of 39% [P. falciparum-infected patients. The understanding of genetic differences in populations substantially transformed the development of vaccines in general and assisted in developing the most recent malaria vaccine [Globally, malaria remains one of the major causes of mortality in children younger than 5 years . Recently of 39% . The divy of 39% . Such di vaccine , 27.IL-22 gene in P. falciparum malaria infection. The aim was to determine whether there is an association in terms of vulnerability or protection as indicated by the level of parasite density or the age group distribution. IL-22 is suggested to have a protective role in severe anemia-associated malaria infection, as it has been reported that IL-22 is elevated in children infected with P. falciparum [P. falciparum malaria; one correlated with protection against SM (rs2227491) and the second with susceptibility to CM (rs2227478) [r2 = 0.92) with rs2227481 and is significantly associated with the risk of P. falciparum malaria in our population. There is a dearth of data to study the role of the IL-22 gene with vulnerability or protection, parasite density and age-related risk of P. falciparum malaria infection.In this study, we examined the frequency of nine SNPs  in the lciparum , 22, 29.2227478) . HoweverIL-22 rs2227481 T allele with protection against P. falciparum malaria infection in a recessive mode of inheritance, with a similar association found for individuals with a homozygous TT genotype. It was also found that the C allele of this SNP is associated with an increased risk of malaria infection. Our highest significant SNP (rs2227481) is in high LD with rs2227476, previously linked to a predisposition to childhood CM in an African population, potentially by altering the binding site for several transcription factors as suggested by the in silico analysis [IL-22 in the clinical manifestation of P. falciparum-associated malaria. It has been reported that the IL-22 rs2227481 polymorphism plays a crucial role in effector T cell pathways [P. falciparum parasite density in the Saudi Arabian population. The rs2227481 T allele was associated with a low parasite density in a dominant model. However, no relationship was observed for an age-related risk of the infection. The results indicate that the rs2227481 T and homozygous TT genotype may be important in protecting against P. falciparum malaria infection.In the present study, we strengthen the possible relationship between the pathways . ExploriP. falciparum infection. Zhang et al. demonstrated that the A and T alleles of IL-22 rs2227483 were significantly elevated in controls compared to tuberculosis (TB) cases and suggested that rs2227483 A and T alleles are associated with increased production of IL-22 decreasing vulnerability to TB infection [P. falciparum infection, the malaria group possessed a higher TT genotype compared to the controls. Our findings and literature suggest that the variant rs2227483 TT genotype could have a role in the altered IL-22 expression and increased risk of malaria infection.The IL-22 variant rs2227483 is located within putative binding sites for the aryl-hydrocarbon receptor (AhR-ARNT) complex, and it has been suggested that this complex transcription factor induce IL-22 expression . The AhRnfection . Notablynfection . AlthougP. falciparum malaria [IL-22 gene and could influence the expression levels of the protein; however, the conclusion should be substantiated by experimental evidence.As parasite density is associated with a more severe outcome in  malaria , 34, we IL-22 gene polymorphism rs2046068 was not statistically associated with either protection or risk of malaria infection or the level of parasite density. Similarly, Weger et al. did not find any relationship of the rs2046068 genotype and haplotype analysis with chronic plaque psoriasis [The soriasis . In the soriasis . We founIL-22 gene demonstrated statistically significant frequencies between the control and P. falciparum-infected malaria groups. We detected a high frequency of the rs1179246-rs1182844-rs976748 A-T-T haplotype in the control group compared to the malaria group, suggesting the role of the A-T-T haplotype in protecting against P. falciparum infection. Similarly, an elevated frequency of the rs2046068-rs2227491 A-G haplotype was observed in the control group. Wang et al. did not find any association between the rs2227491 and serum IL-22 levels of the control group in SLE cases in a Chinese population [P. falciparum-associated clinical outcomes and the function of IL-22 need to be experimentally substantiated.The haplotype distribution analysis at the IL-22 gene and it is also part of the promoter region of the IL-22 gene. Studies suggest that introns are imperative modulators of gene functions such as RNA editing [r2 = 0.98) with rs976748, associated with a higher risk of P. falciparum malaria infection.The variant rs2227513 is positioned at the first intron of the  editing , 38. In P. chabaudi-induced liver damage [Several studies have shown that variations at the IL-22 gene could contribute to the outcome of different diseases. Mastelic et al. demonstrated a shielding role of IL-22 cytokine against r damage . Also, Hr damage . It was r damage . The latr damage . Our finIL-22 gene may produce different roles in the vulnerability to infection in populations with diverse ethnic backgrounds. A plausible explanation of this disparity could be environmental or other genetic elements. As we did not investigate all the IL-22 gene variants, future case control studies are necessary to confirm the findings of this study and to explore additional links between IL-22 SNPs and the clinical outcome of P. falciparum-associated malaria. It is essential to reproduce a similar study in other populations with a different genetic background to replicate our findings in a larger sample size. In addition, studies to outline the molecular or cellular mechanisms underlying the role of IL-22 in malaria are recommended.This study suggests that variants in the P. falciparum malaria. The IL-22 promoter polymorphism in rs2227513 G allele appears to be associated with higher expression levels of IL-22, which could play a role in the protection against the disease. These findings will increase the current understanding of the mechanisms involved in the pathogenesis of malaria infection.Our data shows that the IL-22 rs2227481 TT genotype and T allele and the rs2227483 AT genotype and A allele may be associated with protection against


Triticum kiharae Dorof. et Migush. We first discovered that short peptides derived from the central, N-, and C-terminal regions of one of the WAMPs (WAMP-2) were able to augment the inhibitory effect of Folicur® EC 250, a triazole fungicide, on spore germination of the wheat pathogenic fungi, including Fusarium spp. and Alternaria alternata. In this research, we explored the ability of chemically synthesized WAMP-2-derived peptides for enhancing the sensitivity of two other Fusarium and Alternaria species, F. oxysporum and A. solani, causing wilt and early blight of tomato, respectively, to Folicur®. The synthesized WAMP-2-derived peptides synergistically interacted with the fungicide and significantly increased its efficacy, inhibiting conidial germination at much lower Folicur® concentrations than required for the same efficiency using the fungicide alone. The experiments on co-applications of some of WAMP-2-fragments and the fungicide on tomato leaves and seedlings, which confirmed the results obtained in vitro, are described.There are increasing environmental risks associated with extensive use of fungicides for crop protection. Hence, the use of new approaches using natural plant defense mechanisms, including application of plant antimicrobial peptides (AMPs), is of great interest. Recently, we studied the structural–function relationships between antifungal activity and five hevein-like AMPs from the WAMP (wheat AMP) family of  Solanum lycopersicum L.) is one of the highest-value vegetable crops for the human diet worldwide [Fusarium oxysporum f. sp. lycopersici, and early blight, evoked by Alternaria solani, are the two most destructive for both open-field and greenhouse-cultivated tomatoes [The tomato  and showed they synergistically interacted with tebuconazole to drastically increase the sensitivity of target pathogens to Folicur® EC 250 (25% tebuconazole). The synergism provided higher or complete inhibition of spore germination in these fungi at dosages of Folicur® ineffective alone [® on the conidial germination in F. oxysporum f. sp. lycopersici and to examine the sensitizing potential of WAMP-G1 and WAMP-C towards A. solani. Another important goal of the work was to confirm if in vitro antifungal effects were obtained in two different model plant-pathogen systems. In addition, we studied the antifungal activity of the WAMP-2-derived fragments against A. solani, and compared it with the fragments’ activity against F. oxysporum and the antifungal potency of the native WAMP-2. Thus, this study is a continuation of investigating the novel recently discovered property of hevein-like AMPs to increase the sensitivity of plant pathogenic fungi to agricultural fungicides. The overall goal is to provide a high efficacy of crop protection from diseases while greatly reducing dosages of these antifungal agents.Many properties of antimicrobial peptides (AMPs), such as a wide range of antimicrobial activity  and modet Migush  and othe[Poaceae ,43. Receve alone . The res®EC 250, we estimated the sensitivity of F. oxysporum and A. solani to these fragments. The antifungal activity of the fragments was measured by the level of inhibition of germination of fungal conidia.Before studying the ability of WAMP-2 fragments corresponding to different regions of WAMP-2 to enhance the effect of FolicurF. oxysporum and compared the inhibitory activity of the fragments to each other and to the entire WAMP-2 peptide. Microconidia of the pathogen were treated to each of the WAMP-2 fragments taken at three or four concentrations ranging between 50 and 200 , 100 and 400 (WAMP-G1), or 50 and 400 (WAMP-C) µg/mL. The prior research showed these concentrations were marginally or low toxic for the fungus [F. oxysporum was significantly more sensitive to WAMP-N and WAMP-G2 than to other peptide fragments , the least active fragments against eptides. .F. oxysporum, WAMP-G1 impeded germination of A. solani macroconidia with 50% efficacy at a concentration equal to the ED50 of WAMP-2. At the same time, WAMP-C inhibitory activity against A. solani at the ED50 level significantly exceeded the ED50 of the native peptide for F. oxysporum, and MFC was two times lower than against F. oxysporum.Unlike its inactivity against 50 calculations for Folicur® against A. solani (0.2 ± 0.04 µg/mL) and F. oxysporum (0.2 ± 0.02 µg/mL). It should be noted that the vast majority of known natural and synthetic compounds, like the WAMP-2 fragments, possess an order to several orders of magnitude lower antifungal activity than commercial antimycotics [In general, the synthesized fragments, inhibiting the spore germination by 50% in the range of hundreds or tens of hundreds of micrograms per mL, demonstrated a very low or nominal toxicity towards the target pathogens in comparison to our EDmycotics .® against both fungi. We tested the activity of the fragments, when applied alone, at concentrations that produced marginal (1–9%), low (10–39%), or 40–50% inhibition of the spore germination  significantly exceeded the effects of the fungicide alone. Additionally, in all cases except one (WAMP-G1 100 µg/L and Folicur 0.005 µg/L), Er values exceeded Ee values . These o® was used at the sub-fungicidal dosages, the efficacy of which did not exceed 30% (F. oxysporum) or 45% (A. solani). The assays were performed in 96-well microtiter plates, using a “checkerboard” design. Fungal conidia were treated with either Folicur® or peptide fragments, each alone, and, in parallel, were subjected to combined treatments wherein the fungicide and one of the WAMP-2-derived peptides were mixed in the same concentration as when used alone. In general, such co-applications resulted in a noticeable augmentation of the fungicidal effect against F. oxysporum . In the same range of dosages, the C-terminal fragment co-applied with higher Folicur® concentrations (0.01 or 0.05 µg/mL) enhanced the fungicidal activity more effectively than WAMP-N  [The sensitizing efficacy of WAMP-G2 towards (WAMP-N)  than tha® was co-applied with WAMP-G1 or WAMP-C against A. solani . As in case of F. oxysporum, exposure of conidia to the mixes containing WAMP-C at a concentration of 400 µg/mL provided almost complete suppression of A. solani germination.Co-application of WAMP-C and the fungicide against ergistic b. The reA. solani and F. oxysporum to the fungicide by the synthesized peptide fragments might be subsequently realized on plants, we determined the effectiveness of the sensitization of these two pathogens to Folicur using live tomato tissue assays. These experiments were performed using detached tomato seedlings and leaf disks treated with combinations of Folicur® mixed with WAMP-2-derived oligopeptides. For these experiments, WAMP-C and WAMP-G1, which showed different antifungal and sensitizing activities towards these two pathogens under in vitro conditions, were chosen. The C-terminal oligopeptide was the most active sensitizer of F. oxysporum compared to all other fragments, while WAMP-G1 combined with the fungicide provided mainly the synergistic effect towards A. solani.In order to confirm that the sensitization of F. oxysporum, a conidial suspension was prepared and divided into three portions. One portion was diluted with an equal volume of SDW to serve as a pathogen-inoculated control. The second portion was mixed with an equal volume of aquatic Folicur®, and the third portion was added to the fungicide combined with either WAMP-C or WAMP-G1. Final concentrations in each portion included microconidia at 3 × 106/mL, peptide fragments at 200 µg/mL, and Folicur® at two concentrations (0.005 and 0.05 µg/mL). Stems of tomato seedlings with four or five true leaves were detached from roots and placed in the prepared suspensions for two days, so that only stems, but not leaves, remained submerged. Inoculated seedlings were incubated with the detached seedlings in Knop nutrient solution for the next three post-inoculation days (PIDs). To confirm that WAMP-derived peptides possessed no phytotoxicity, two other groups of seedlings were preliminary incubated in SDW or that added with the peptides. These preliminary assays showed WAMP-C and WAMP-G1 had no phytotoxicity at the used concentration  throughout the observation period  did not differ from that in the control (Drops of the test suspensions containing Folicur/peptide-treated or SDW-treated (control) conidia were applied to the adaxial side of leaf disks placed on the surface of water agar in Petri plates. After inoculation, the plates were transferred to a climate chamber for disease development. At the 9th day of incubation, the damage symptoms on leaf disks were scored. The lesions on control disks developed beyond inoculation drops. Almost the entire surface yellowed on three of the six treated disks. Similar symptoms were also observed on leaf disks exposed to Folicur control a–c. In c control d.F. culmorum but was able to suppress the spore germination in F. oxysporum and A. alternata. We also found that the terminal  fragments and the oligopeptides derived from the central region of the WAMP-2 molecule  significantly enhanced the sensitivity of some soil-borne and foliar cereal-damaging fungi to a triazole fungicide, tebuconazole, formulated as Folucur®EC 250 [Along with the structural and functional similarities with other hevein-like AMPs, WAMPs have a number of specific characteristics ,46, dispr®EC 250 , while WF. oxysporum and A. solani to Folicur with the aforementioned WAMP-2-derived oligopeptides in two model plant-pathogen systems. The obtained results confirmed our previous findings. All tested oligopeptides augmented the inhibitory efficiency of the fungicide towards conidial germination in these fungi. Results also showed, in general, the sensitizing activity of synthesized N- and C-terminal fragments was greater compared to the sensitizing activity of peptides from the central region of WAMP-2  . Folicur05 µg/mL . The resr WAMP-C , confirmsc assay . Since pF. graminearum in vitro and under greenhouse or field conditions. In addition, interaction with fungicides resulting in synergistic reduction of biofilm formation in Candida albicans was shown for radish defensins [In spite of quite limited studies related to chemosensitizaton of pathogenic fungi to agricultural fungicides using AMPs ,29,44,48efensins .®, first discovered in our previous study and also revealed in this work, is evidently attributed to the fact that these sensitizing agents attack different targets in the tested plant pathogens. This resulted in a severe weakening of the pathogen vitality and a drastic enhancement of the fungicidal effect.Triazoles, belonging to DMI fungicides, inhibit 14-α-demethylase from participating in the synthesis of ergosterol, the principal sterol in fungal cell membranes . On the It should be mentioned that triazole fungicides control the development of various plant pathogenic fungi by effective suppression of their growth. It is commonly held that they are unable to inhibit germination of their spores with the same efficacy, since fungal spores already contain ergosterol. However, our results suggest that combining triazoles with WAMP-2 fragments may increase the efficiency of these fungicides against spore germination, the key stage of the plant infection process.A further factor to consider, in addition to enhancing the fungicidal effect using WAMP-2 fragments, is the potential for reducing the effective dosage levels of triazoles required for fungal control. This could mitigate pesticide impact on the environment. Furthermore, resistant strains pathogenic to tomato will probably be better controlled with triazoles without increasing their dosages because of significant enhancement of the fungicidal effect by the addition of the sensitizing peptides.However, so far, our results show only the potential for the synthesized WAMP-2 fragments to enhance the effectiveness of industrial fungicides in agricultural practice. These results should be confirmed not only in plant-pathogen model systems but also by experiments with plant treatments. As compared to recombinant WAMP-2, commercial production of which would be complicated, obtaining short peptides via direct chemical synthesis is practicable. Testing the sensitizing activity of other plant AMPs, and their fragments, is necessary to select the most active sensitizers and to understand the interrelationship between the peptide structure and sensitizing activity. In addition, such investigations could contribute to our knowledge of chemosensitization mechanisms. Another important issue is study of the range of action of the revealed sensitizers. Triazole fungicides giving the most effective combinations with WAMP-2 fragments, and crop pathogens most responsive to the combined treatments should be revealed using various plant-pathogen model systems, and also under greenhouse and field conditions.Fusarium oxysporum f. sp. lycopersici (Sacc.) W.C. Snyder and H.N. Hans (strain F37), causing wilt of tomato, was obtained from the curated collection of plant pathogens of the Laboratory of Physiological Plant Pathology at the All-Russian Research Institute of Phytopathology (ARRIP). Alternaria solani (Ell. & Mart.) L.R. Jones and Grout. (strain MO-VNIIF-9-2018), a causative agent of tomato early blight, isolated from naturally infected plants cultivated on the ARRIP experimental field in the Moscow region, was provided by the ARRIP Department of Potato and Vegetable Diseases. Stock cultures of the pathogenic strains were maintained on potato dextrose agar (PDA) slants in the dark at 25–26°C by sub-culturing after each four-six weeks.F. oxysporum) or 14 (A. solani) days. Fungal conidia were collected from the colony surfaces and separated from mycelial debris, as described earlier [A. solani was additionally stimulated by flooding mycelia with sterilized ice water followed by short-time UV-irradiation [To obtain spore-producing colonies and prepare spore suspensions, the pathogens were re-cultured on PDA in 9-cm Petri plates under the above conditions for 7  and purified by HPLC on a Luna C8 column  as described earlier [Four peptides corresponding to N- and C-terminals, as well as central regions of the peptide WAMP-2 , were pr earlier ,52. MALD earlier .Freeze-dried peptide preparations were stored at −20°C and dissolved in sterilized distilled water (SDW) on the day of testing their antifungal or sensitizing activities.E. coli BL21 (DE3), as described earlier [To compare the antifungal activity of synthetic fragments with that of WAMP-2, WAMP-2 recombinant peptide was obtained as fusion protein with thioredoxin in  earlier ,51. The ® EC 250, a commercial pesticide formulation , containing 25% tebuconazole as the active ingredient, was used for both in vitro and in planta experiments. Daily-fresh Folicur® solution in SDW with final concentrations ranging from 0.005 to 0.05 µg/mL and from 0.01 to 0.2 µg/mL in the case of F. oxysporum and A. solani, respectively, were prepared prior to mixing with the WAMP-2 fragments or application on tomato leaf disks and seedlings. For both fungi, treatment at these concentrations, revealed in preliminary experiments using serial double or fivefold dilutions, resulted in a sub-fungicidal effect, not exceeding 30–45%.Folicur5 microconidia/mL (F. oxysporum) or 3 × 104 macroconidia/mL (A. solani). Resulting suspensions were incubated at 20–22 °C overnight in wells of 96-well microtiter plates. Control suspensions  were prepared using SDW. Thereafter, germinated and non-germinated conidia (500 ones of each pathogen per treatment) were counted in treated and control suspensions using an inverted microscope. The average number of conidia germinated in these suspensions was calculated and expressed as the percent of the total number of the counted conidia. In addition, percent germination inhibition in the treated conidia, relative to germination in controls, was determined and indicated as levels of the inhibitory effect in Fusarium and Alternaria species [The inhibitory effect of synthesized peptides on conidial germination of fungi was assessed by a microtiterplate assay . Fungal  species .® EC 250 were designed under the principle of a microdilution checkerboard assay [® or the oligopeptides, alone, used at the same final concentrations as in the mixtures.In vitro tests involving pathogen sensitization to Folicurrd assay . Fungal F. oxysporum was determined earlier under the same experimental conditions [WAMP-2 antifungal activity against nditions ,44.Solanum lycopersicum L.) seeds of cv. Bely Naliv, a variety susceptible to Fusarium wilt, were surface-disinfected, germinated, and sown in disinfected sand. Seedlings were grown to the stage of 4–5 true leaves as described earlier [F. oxysporum at a final concentration of 3 × 106 microconidia/mL. Other portions of the detached seedlings were submerged into vials with an equivalent conidial suspension containing an aquatic solution of Folicur® (0.05 µg/mL) alone, or combined with either WAMP-C or WAMP-G1 . Control detached seedlings were placed in SDW. The seedlings were incubated in a climate chamber at 27–28°C, 150 µEm−2 s−1 (16-h photoperiod). After two-day inoculation with the pathogens, seedlings were transferred into 25% Knop solution that was refreshed daily. Wilting symptoms were observed every day for five days and photographed at 1st, 3rd, and 5th post-inoculation days (PID).Tomato  was mixed with 1 mL of WAMP-G1 (400 μg/mL), Folicur® (0.1 μg/mL), or the fungicide combined with WAMP2-G1 (each at the indicated concentration). The resulting test mixes contained fungal spores, WAMP-G1, and Folicur® at the final concentrations of 2.5 × 104 conidia/mL, 200 µg/mL, and 0.05 µg/mL, respectively. A sample of the suspension in SDW with the same final concentration of conidia was used as a control. After 1-h incubation at 23–24°C, 50-μL aliquots of the treated mixes and control suspensions were pipetted onto the adaxial side of a leaf disk. The plates with inoculated leaf disks were transferred to a climate chamber and incubated as described (see Leaves of 6-week-old tomato plants (cv. Volgogradsky) grown as described above  were wasibed see . Early b50, and ED99, respectively) were determined by the probit analysis [Y = ax + b) used in probit analyses, Y is the probit value for 50% or 99% inhibition levels, x values are decimal logarithms of the correspondent concentrations, and a and b represent regression coefficients. The calculated ED99 values from the probit analysis were considered as the minimum fungicidal concentration (MFC), i.e., the lowest concentration of a peptide, used alone, resulting in 99.9% inhibition of conidial germination.Inhibitory concentrations of the peptides providing in vitro 50% or 99% in vitro suppression of conidial germination . Experiments evaluating the sensitizing activity of the tested peptides on leaf disks and detached tomato seedlings were additionally repeated twice. Mean values, standard deviations, standard errors, as well as significant differences from controls and between treatments , were calculated using STATISTICA v. 6.1 software . Approximation of confidence values (R2) and regression coefficients were determined using statistical functions of Microsoft Excel 2013 .For each of the two pathogens, microtiter plate tests with synthesized peptides included three independent experimental series. Each experiment included 5 replications of each treatment, 100 conidia per each one (


However, no consensus has been reached on a standardised geometry. The CF is dependent on the selected geometry and is further affected by partial volume effects. This study investigated the effect of two CF geometries and their corresponding recovery coefficients (RCs) on the quantification accuracy of The CF geometries investigated were (i) a radioactive-sphere surrounded by non-radioactive water (sphere-CF) and (ii) a cylindrical phantom uniformly filled with radioactive water (cylinder-CF). Recovery coefficients were obtained using the sphere-CF and cylinder-CF, yielding the sphere-RC and cylinder-RC values, respectively, for partial volume correction (PVC). The quantification accuracy was evaluated using four different-sized spheres (15.6–65.4 ml) and a kidney model with known activity concentrations inside a cylindrical, torso and patient phantom. Images were reconstructed with the 3D OS-EM algorithm incorporating attenuation, scatter and detector-response corrections. Segmentation was performed using the physical size and a small cylindrical volume inside the cylinder for the sphere-CF and cylinder-CF, respectively.The sphere quantification error (without PVC) was better for the sphere-CF (≤ − 5.54%) compared to the cylinder-CF (≤ − 20.90%), attributed to the similar geometry of the quantified and CF spheres. Partial volume correction yielded comparable results for the sphere-CF-RC (≤ 3.47%) and cylinder-CF-RC (≤ 3.53%). The accuracy of the kidney quantification was poorer (≤ 22.34%) for the sphere-CF without PVC compared to the cylinder-CF (≤ 2.44%). With PVC, the kidney quantification results improved and compared well for the sphere-CF-RC (≤ 3.50%) and the cylinder-CF-RC (≤ 3.45%).The study demonstrated that upon careful selection of CF-RC combinations, comparable quantification errors (≤ 3.53%) were obtained between the sphere-CF-RC and cylinder-CF-RC, when all corrections were applied. The availability of the co-registered SPECT and CT data has made routine attenuation compensation practical and easy to implement. Multiple window scatter compensation methods such as the dual energy window (DEW) and the triple energy window (TEW) methods are available with most gamma camera vendors, and these methods can be used to apply scatter correction during or post-reconstruction. However, model-based scatter correction methods, such as effective source scatter estimation (ESSE), model the scatter function more accurately and thus better resemble the true scatter distribution . The ESS phantom  and exte phantom . Another phantom .−3). Thus, a calibration factor (CF) has to be applied to obtain SPECT reconstructed images in units of radioactive concentration. It is only recently, with modern SPECT/CT systems, that reconstructed SPECT images are routinely provided in units of radioactive concentration. An example of such a SPECT/CT system is the Siemens Symbia Intevo™ scanner , which incorporates a point source traceable to a secondary standard laboratory for calibration purposes and conversion of the reconstructed SPECT images to activity concentration.SPECT was traditionally regarded as a non-quantitative imaging modality, unlike its counterpart PET. In both modalities, advances in hybrid systems include automation of compensation methods for photon attenuation, scatter and limited spatial resolution in a unified manner using iterative reconstruction algorithms. In general, SPECT images are obtained in units of image counts, unlike PET reconstructed images, which have units of tissue radioactive concentration (kBq.cm177Lu SPECT quantitative imaging [177LuCl3. The point source in air and the sphere in water yielded the worst and best results respectively for the activity quantification in an anthropomorphic torso phantom. The sphere in a non-radioactive background was reported as suitable for activity quantification of 177Lu SPECT data using the 208 keV photopeak. Superior quantification accuracy has been reported for SPECT imaging with a CF obtained using a sphere in a radioactive background (sphere-to-background ratio of 6:1) compared to that obtained using a cylindrical phantom uniformly filled with 131I [131I and 188Re, Zhao et al. [177Lu 208 keV photopeak. In addition, the study showed that CFs obtained using spheres in a non-radioactive background may overestimate the CF by 10% attributed to the underestimation of the TEW scatter approximation and accentuated further by the attenuation correction during reconstruction.The system sensitivity, as defined by the National Electrical Manufacturers Association (NEMA) , is conv imaging . These aith 131I . The auto et al.  suggeste177Lu SPECT activity quantification accuracy reported by different authors for various phantoms, as well as their data acquisition and processing protocols, have been summarised in the MIRD pamphlet 26 [177Lu SPECT quantitative imaging established using the Simulating Medical Imaging Nuclear Detectors (SIMIND) MC program [177Lu and 131I. These phantom geometries included a sphere placed centrally in air and in water as well as at an offset of 8.6 cm in water. The authors concluded that none of their geometries were sufficient to be used individually and recommended that a mean value obtained from the different geometries should be incorporated. They also suggested that the characterisation of the change in the CF with lesion volume was necessary due to the PVE and have proposed that the CF and RC should be combined in a mean calibration coefficient. This approach may require that a large range of CF values be determined from different geometries and tabulated. In addition, the CF would be affected by the VOI definition and further affected by the segmentation method used to determine the RC. In another publication, Wevrett et al. [177Lu SPECT quantitative imaging.The disparities between phlet 26 . The MIR program . However program  demonstrt et al.  assessed177Lu SPECT images using MC simulations. The CFs were obtained from (i) a radioactive sphere surrounded by non-radioactive water, termed sphere-CF, and (ii) a cylindrical phantom uniformly filled with radioactive water, termed a cylinder-CF. Two sets of RC curves were generated as a function of object size, using the two CFs. The first RC curve was constructed using the sphere-CF and the second RC curve was created using the cylinder-CF; the two curves were used to obtain the sphere-RC and cylinder-RC values, respectively. The effects of the sphere-CF-RC and cylinder-CF-RC combinations, on the quantification accuracy of 177Lu activity derived from SPECT images, were evaluated using three phantom geometries.This study aimed to investigate the effect of two CF geometries and their corresponding RCs on the quantification accuracy of 177Lu SPECT images with a model of a Siemens Symbia T16 hybrid SPECT/CT (Symbia T16)  dual-head gamma camera was validated in a previous study [The SIMIND MC program (SIMIND version 6.1.2) was used in this study. Its ability to successfully mimic us study . The valus study . The difus study  as descrus study . The thr177Lu using a medium-energy (ME) collimator with a 20% energy window centred over the 208 keV photopeak (187.2–228.8 keV) [The simulations were set up for 8.8 keV) , 49–51. 8.8 keV) . All sim8.8 keV) , 52.first phase of the study focused on three essential steps required to optimize the reconstruction parameters and the quantification process. The steps consisted of (i) optimizing the 3D OS-EM reconstruction algorithm, (ii) establishing the CFs and (iii) determining the RCs. The second phase of the study evaluated the quantification accuracy, reported as the quantification error, achieved with the steps established in the initial phase by quantifying spherical inserts in the three above-mentioned phantoms. The kidney quantification in the patient phantom was also evaluated. The simulations of the phantoms comprised of simulated projection images and result files describing the simulation protocol. A 3D OS-EM algorithm was used for image reconstruction and employed CT-based attenuation correction, ESSE scatter correction [Our study comprised of two phases. The rrection , 53 and rrection . Sphericrrection , 55, 56.177Lu concentration of 3.04 MBq/ml. This is comparable to concentrations reported in the literature for similar investigations [For the first phase of the study, each of the phantom geometries in the quantification steps was simulated with a uniform igations . DiffereTo determine the optimal number of 3D OS-EM updates required for convergence (where 90% of the true activity is recovered), SPECT images of a cylindrical phantom equipped with radioactive spheres with volumes (and their corresponding diameters) of 4.2 ml (2.0 cm), 14.1 ml (3.0 cm) and 65.4 ml (5.0 cm) in a warm background were simulated. To further investigate the effect of the sphere-to-background ratio on the convergence, each sphere was simulated using sphere-to-background ratios of 6:1 and 13:1 , 57, 58.177Lu 208 keV photopeak using a ME collimator was determined in our previous study, resulting in a FWHM of 1.1 cm [177Lu is a costly therapeutic isotope, with a relatively long physical half-life of 6.7 days, the non-radioactive background of the sphere-CF offered a more practical alternative that could easily be implemented in clinical practice compared to the cylinder-CF. The CFs were calculated using the general formula shown in Eq. V is the volume of the VOI and TC is the true concentration obtained from the SIMIND result file. For the cylinder-CF, the cylindrical VOI was defined in a uniform area in the centre of the image to exclude edge effects, as recommended by D’Arienzo et al. [The sphere-CF was calculated using a simulation of a 65.4 ml (5.0 cm) radioactive sphere placed in the cylindrical phantom Fig. a with a f 1.1 cm . The cylo et al. . The VOI177Lu and radioactive spheres of eight varying sizes below and above the known system spatial resolution, with known concentration. The sphere volumes (and diameters) used were 2.4 ml (1.5 cm), 4.2 ml (2.0 cm), 8.2 ml (2.5 cm), 14.1 ml (3.0 cm), 22.4 ml (3.5 cm), 33.5 ml (4.0 cm), 47.7 ml (4.5 cm) and 65.4 ml (5.0 cm) with a sphere-to-background ratio of 6:1. The concentration was calculated using the sphere-CF and the cylinder-CF and expressed as a fraction of the true concentration (Eq. RC is the recovery coefficient and CSPECT and Ctrue are the SPECT estimated and true activity concentrations (MBq/ml) in the spheres, respectively. The values of the coefficients from the curve function shown in Eq. y indicates the required RC value, x is the sphere diameter and a, b, and c are the fitting constants. The application of sphere-based RC curves (look-up tables) for PVC of the kidneys based solely on volume dependence has been reported to be non-optimal for 177Lu SPECT/CT image quantification [The RCs for partial volume compensation were obtained from simulated SPECT images of the cylindrical phantom uniformly filled with  14.1 ml .0 cm, 22fication . This wa 14.1 ml .0 cm, 22177Lu SPECT quantification error in the three phantoms. The objective of the first phantom  was to determine the 177Lu quantitative error of the spherical inserts in a phantom that characterised a simple cylindrical geometry and a homogeneous attenuating medium. The second phantom (torso phantom) represented a more complex geometry as well as a non-homogeneous attenuating medium. The third phantom extended the quantification error to a clinically realistic patient phantom. All the phantoms were simulated four times with the inclusion of one of the spheres of volume: 15.6 ml (3.1 cm), 24.4 ml (3.6 cm), 33.5 ml (4.0 cm) and 65.4 ml (5.0 cm) for each simulation, respectively. The sphere concentrations were 2.2 MBq/ml with a sphere-to-background ratio of 13:1. The sphere-to-background ratios selected for the three phantom simulations were different from those simulated for the RC (6:1) to avoid biased results in the determination of the quantification error. Each sphere was simulated in the centre of the cylindrical phantom as well as in the abdomen next to the liver for the torso and patient phantoms. The relative concentration in the various structures found in these two phantoms was defined to reflect typical activity distribution of 177Lu-DOTATATE [177Lu-DOTATATE pharmacokinetic study [The second phase of the study evaluated the DOTATATE , 59. Theic study .Two sets of quantification results were obtained for each of the phantoms. Firstly, the counts were converted to concentration using the sphere-CF without PVC to generate the sphere-CF quantification error, and subsequently corrected for partial volume using the corresponding sphere-RC to generate a sphere-CF-RC quantification error. Secondly, the counts were converted to concentration using the cylinder-CF without PVC to obtain the cylinder-CF results and the cylinder-RC applied for PVC, resulting in the cylinder-CF-RC data. The quantification error was calculated as the percentage difference between the true concentration (true (MBq/ml)) used as input into SIMIND and the quantitative SPECT estimated concentration (SPECT (MBq/ml)) calculated from the images Eq. .5\docum177Lu SPECT images of three different-sized spheres simulated in a cylindrical phantom, as well as the left and right kidneys of the patient phantom. The images used to calculate these values were derived from simulations when the object-to-background ratios of 13:1  were the same for all VOIs, the smaller spheres had a lower SPECT estimated concentration (CSPECT) values, demonstrating the PVE. It is evident from these curves that RC is strongly dependent on the size of the spheres. The fit of the curves in Fig. Figure 0.99 Eq. , generatThe sphere-RC and cylinder-RC for the spheres and kidneys are summarised in Table Table As seen in Table The quantitative error results obtained for the spheres simulated in the torso phantom are demonstrated in Table Without PVC, the quantification error showed an average underestimation of 4.50 ± 1.24% for the sphere-CF Table . SimilarIt can be seen from the results in Table Figure As seen from Table ) obtained for the cylinder-RC. This was contrary to the sphere quantification results where a considerable underestimation of the quantification error was obtained using the cylinder-CF without PVC. The kidney quantification error improved to 2.26 ± 1.76% with the sphere-CF-RC corrections. With PVC the sphere-CF-RC and cylinder-CF-RC yielded comparable average quantification results of 2.26 ± 1.76% and 2.20 ± 1.77%, respectively; similar to the findings of the sphere quantification results. The results suggest that PVC did not play a major role in the kidney quantification error using the cylinder data. This may be attributed to the fact that the cylinder-RC value applied to the kidneys was close to 1.0.The kidney quantification showed an average overestimation of 20.66 ± 2.38% for the sphere-CF without PVC compared to the better results  gamma cameras, similar to that simulated in the current study, have been reported in the literature. The reported CFs were all obtained for a 0.95-cm-thick NaI (Tl) detector crystal equipped with a ME collimator using a 20% energy window centred over the 208 keV photopeak. The reconstructed images were all corrected for attenuation, scatter and CDR; however, the correction methodologies varied. Although the CFs were acquired with similar gamma camera settings, the values differ vastly depending on the phantom configurations, VOI definition, the gamma camera used, and the types of corrections applied to the data. A value of 20.2 cps/MBq for a cylindrical phantom with a VOI containing the entire phantom is reported for a Siemens Symbia Intevo Bold SPECT/CT system  [The underestimation of the sphere-CF in comparison to the cylinder-CF value was attributed to PVE due to spill-out of counts. Some authors have suggested the simple use of an additional 3 cm margin to the physical dimensions of the sphere to include spilled-out counts . Other aGermany) . The samGermany) . While oGermany) . The aboCreating sphere-RC and cylinder-RC curves aimed to obtain approximate RC values for spherical objects of different sizes where spill-out of counts were present and when PVC is required. In order to use sphere-RC and cylinder-RC curves from Fig. 99mTc and 177Lu SPECT studies [The sphere quantification error results Table  obtained studies . This ty studies , 32. The177Lu SPECT activity quantification of radioactive spherical inserts placed in a cylindrical phantom with a radioactive background. The authors applied the ESSE scatter correction method and compensated for PVEs by drawing VOIs larger than the spherical inserts and reported errors of 10% for their largest sphere of 37 ml. Sanders et al. [The quantification errors obtained in this study compared well with literature investigations of similar phantom and patient geometries, which employed OS-EM-based reconstruction algorithms with compensation for scatter, attenuation using CT data and CDR. De Nijs et al.  investigs et al.  reporteds et al. . The aut177Lu SPECT activity quantification of spheres measured in the same torso RSD phantom, as simulated in our study, and reported errors of 15% for their largest spherical insert (104 ml). They used an accelerated MC simulation method for scatter correction and CT-based VOIs without PVC. The quantification errors found in our study (≤ − 1.43 ± 3.01%) were comparable to that of D’Arienzo et al. [Hippeläinen et al.  investigo et al.  who quano et al.  applied o et al.  used a t177Lu SPECT activity data of five patients acquired with the Symbia TruePoint T6 SPECT/CT gamma camera . The authors evaluated their quantification error by comparing the calibrated activity injected into the patients with the quantified activity obtained by drawing a VOI surrounding the patient using a − 400 Hounsfield unit threshold for the CT images. Although their quantification method was different from ours, their results were comparable to our patient phantom findings, when all corrections were applied, with sphere and kidney quantification errors of ≤ 2.20 ± 0.94% and ≤ 2.26 ± 1.76%, respectively. Sanders et al. [177Lu bladder activity concentrations obtained from SPECT/CT patient images with urine sample measurements from calibrated well counters. Their segmentation method for the bladder included placing a smaller ellipsoid VOI in the bladder’s interior to avoid edge roll-off that causes variations in the iso-contour measurements. The total mean counts in the VOI were normalised to the volume of the bladder. The method yielded mean quantification errors of 10 ± 8.3%. Bailey et al. [177Lu activity quantification of whole body planar scans and cardiac blood pool SPECT images. Hippeläinen et al. [177Lu-DOTATATE treatment using SPECT images. The systematically higher activity quantification errors were attributed to CT-based VOIs that were sensitive to PVEs and estimates obtained at different time points. Willowson et al. [177Lu SPECT/CT using geometry specific RCs for PVC was investigated using a 3D-printed two-compartment kidney phantom [Beauregard et al.  reporteds et al.  comparedy et al.  reportedn et al.  reportedn et al.  investig phantom . The bes177Lu SPECT imaging of voxelized phantoms as validated by Ramonaheng et al. [177Lu SPECT quantification errors in the three phantoms were comparable for the sphere-CF-RC and cylinder-CF-RC combinations. Our absolute quantification errors of smaller than and equal to 3.53% for the three phantoms, compared well to literature findings and complied with the ± 5% absorbed dose accuracy required for molecular radiotherapy [177Lu SPECT activity quantification upon the careful selection of a CF-RC combination, certain considerations may be limiting. These include firstly, that the application of patient-specific RCs in the clinic entails the use of CT or MRI data in combination with MC simulations. This would require modelling of CF-RC combinations for every patient geometry and activity distribution, which may be cumbersome to implement routinely. Secondly, the presence of non-uniform activity distributions may further complicate activity quantification. Thirdly, the segmented kidney volumes used in this study were obtained from low-dose, low-resolution, non-contrast-enhanced CT data, which was inadequate to differentiate between the different kidney compartments, such as the medulla, cortex and renal pelvis, reliably. For this reason, each whole left and right kidney was assigned a uniform activity concentration. High-resolution image data such as contrast-enhanced CT images or MRI would facilitate segmentation and VOI delineation that is more reliable with a better representation of a clinical kidney model. Another limitation of this study is the methodology used to calculate the kidney RCs directly from Eq. In this study, we used the SIMIND MC program to model a Siemens Symbia T16 gamma camera for g et al. . Emanatiotherapy . Althoug


Objectives: The aim of this study was to assess the diagnostic role of eosinophils count in COVID-19 patients. Methods: Retrospective analysis of patients admitted to our hospital with suspicion of COVID-19. Demographic, clinical and laboratory data were collected on admission. Eosinopenia was defined as eosinophils < 100 cells/mm3. The outcomes of this study were the association between eosinophils count on admission and positive real-time reverse transcription polymerase chain reaction (rRT-PCR) test and with suggestive chest computerized tomography (CT) of COVID-19 pneumonia. Results: A total of 174 patients was studied. Of those, 54% had positive rRT-PCR for SARS-CoV-2. A chest CT-scan was performed in 145 patients; 71% showed suggestive findings of COVID-19. Eosinophils on admission had a high predictive accuracy for positive rRT-PCR and suggestive chest CT-scan  and 0.84 (95% CIs 0.77–0.91), respectively). Eosinopenia and high LDH were independent predictors of positive rRT-PCR, whereas eosinopenia, high body mass index and hypertension were predictors for suggestive CT-scan findings. Conclusions: Eosinopenia on admission could predict positive rRT-PCR test or suggestive chest CT-scan for COVID-19. This laboratory finding could help to identify patients at high-risk of COVID-19 in the setting where gold standard diagnostic methods are not available. At the end of December 2019, the first case of COVID-19 (Coronavirus disease 2019) caused by a novel human coronavirus, called SARS-CoV-2, was reported in Wuhan, China . COVID-1On 15 April 2020 more than two million COVID-19 cases were confirmed in more than 100 countries and over 130,000 deaths declared ,7. In syDue to the high contagious power of the virus, rapid and accurate diagnostic tools are required to rapidly identify potentially infected patients; this would avoid further viral diffusion in the population and help to adequately manage patients with moderate or severe symptoms. The gold standard for COVID-19 diagnosis is based on real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) test from nasopharyngeal swab specimens or, in severe cases, from broncho-alveolar lavage (BAL) , chest cNevertheless, rRT-PCR requires laboratory facilities and provides results with a delay ranging from few hours to some days. In addition, chest CT-scan is not easily available in all hospitals worldwide and has additional issues, such as costs and the exposure to ionizing radiations. As such, it could be interesting to evaluate whether commonly available laboratory data, combined with clinical evaluation, could help physicians to identify patients at high-risk for COVID-19 diagnosis, in particular in areas with lack of resources. In a small study, eosinopenia and lymphopenia on admission were reported as useful indicators of COVID-19 in patients with typical symptoms and radiological abnormalities . Considering the scarce data on this topic, the aim of this study was to assess the potential role of eosinophils count to predict positive rRT-PCR test or suggestive chest CT-scan findings in patients admitted for suspected COVID-19.This monocentric retrospective cohort study was performed in the COVID departments of the Hôpital Erasme in Brussels, Belgium. The study protocol was approved by the local ethics committee (P2020/246), which waived the need for informed written consent because of its retrospective design and since all interventions were part of the standard patients’ care. None of these patients has been included in previously published studies on this topic.We enrolled patients admitted to our hospital from March 10  to 31 March 2020 with the following inclusion criteria: (a) age of 18 years or older; (b) tested using rRT-PCR assay on the nasopharyngeal swab and/or BAL specimens for suspicion of COVID-19; (c) available laboratory exams on the admission to the hospital. Exclusion criteria were patients already admitted in our institution for other reasons  and patients transferred from other hospitals. 3.We collected demographics, comorbidities, patients’ characteristics on admission as well as results of rRT-PCR and chest thin-section CT-scan. Chest CT evaluation was performed by one radiologist (PAG), who reported whether the CT-scan was suggestive or not for COVID-19  ,13. For The primary endpoint of this study was to assess the association of eosinophils count on admission and a positive rRT-PCR test. Secondary outcomes included the association of eosinophils count on admission and a suggestive chest CT-scan and the identification of predictors of positive rRT-PCR test and suggestive CT-scan. In addition, we assessed whether eosinopenia was able to predict disease severity on admission, according to WHO criteria  .t-test or Mann–Whitney U-test for continuous variable, as appropriate. The discriminative ability of eosinophils to predict a positive rRT-PCR or a suggestive CT-scan was evaluated using receiver operating characteristic (ROC) curves with the corresponding area under the curve (AUROC). Youden’s index was computed to assess the optimal cut-off of eosinophils for sensitivity and specificity to predict positive rRT-PCR or suspected chest CT-scan findings. Multivariable logistic regression analysis with positive rRT-PCR as the dependent variable was performed in all patients. Collinearity between variables  was excluded prior to modelling; only variables associated with positive rRT-PCR in the univariate analysis (p < 0.1) were included in the multivariate model. Odds ratios (OR) with 95% confidence intervals (CI) were computed. A similar approach was used to perform the multivariate analysis with suspected CT-scan as the dependent variable. A p < 0.05 was considered as statistically significant. Statistical analyses were performed using the SPSS 25.0 for Macintosh .Descriptive statistics were computed for all study variables. A Kolmogorov–Smirnov test was used, and histograms and normal-quantile plots were examined to verify the normality of distribution of continuous variables. Discrete variables were expressed as count (percentage) and continuous variables as mean ± SD or median (25th–75th percentiles), as appropriate. Demographics and clinical differences between groups  were assessed using a chi-square test or Fisher’s exact test for categorical variable, and Student’s The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Of the 264 patients fulfilling the inclusion criteria, 90 patients were excluded as already hospitalized or transferred from other hospitals ; as such3 and 134/174 (77%) patients presented eosinopenia. Eosinophils were significantly lower in patients with positive rRT-PCR than others . Patients with anosmia also had more frequently eosinopenia than others, although this was not statistically significant (Eosinophils count on admission was 10 [0–30]/mmnificant .p < 0.01—3 for the best combination of sensitivity (94%) and specificity (63%) to predict positive rRT-PCR. The highest positive predictive value  was observed for an eosinophils count < 5/mm3, which was associated with a specificity of 86%, a sensitivity of 59% and a negative predictive value (NPV) of 64%. The highest NPV  was observed for an eosinophils count > 100/mm3, which was associated with a specificity of 44%, a sensitivity of 95% and a PPV of 66%. In the multivariate analysis, eosinopenia and elevated LDH were independent predictors of positive rRT-PCR (OR 13.42 (95% CIs 4.11–43.77) p < 0.01 and OR 1.01 95% [CIs 1.00–1.01] p = 0.02, respectively) (Table 3). The eosinophils count on admission was similar between patients with mild/moderate and severe/critical COVID-19 .Patients with positive rRT-PCR had a higher BMI, higher values of CRP, PT and LDH and lower white blood cells count, albumin levels and lymphocyte to CRP ratio than others . In partp < 0.01, p < 0.01).Eosinophils count on admission was significantly lower in patients with suggestive chest CT-scan than in others (20 (0–20) vs. 95 (20–170); p < 0.01; OR 16.46 (95% CIs 6.49–41.77), p < 0.01) and higher levels of CRP, LDH, PT, fibrinogen and LCR, as well as lower albumin levels. As for RT-PCR, all subtypes of white blood cells were significantly lower in patients with a suggestive chest CT-scan when compared to others. Predictive accuracy of white blood cells subtypes to predict a suggestive chest CT-scan is shown in p < 0.01, 3 for the best combination of sensitivity (82%) and specificity (74%) to predict a suggestive chest CT-scan. The highest positive predictive value  was observed for an eosinophils count <5/mm3, which was associated with a specificity of 93%, a sensitivity of 55% and a NPV of 46%. The highest NPV  was observed for an eosinophils count >100/mm3, which was associated with a specificity of 45%, a sensitivity of 91% and a PPV of 80%. In the multivariate analysis, high BMI (OR 1.09 [95% CIs 1.01–1.19] p = 0.04), arterial hypertension (OR 3.30 [95% CIs 1.29–8.46] p = 0.01) and eosinopenia (OR 8.12 [95% CIs 2.61–25.23] p < 0.01) were independent predictors of suggestive chest CT-scan for COVID-19  vs 7/42, 17%), COVID-19 . In this study, eosinopenia on admission was a common finding in patients with a positive rRT-PCR as well as those with a suggestive chest CT-scan for COVID-19. Eosinophils count had a high predictive accuracy value and was independently associated with both positive rRT-PCR and suggestive chest CT-scan. As such, the combination of eosinophils count on admission and other clinically relevant variables may help physicians to identify patients at risk of COVID-19 if laboratory or imaging resources are lacking.Eosinophils are circulating and tissue-resident leukocytes, which show proinflammatory, immunoregulatory and antiviral effects . Eosinop3) on admission13; similar results were also reported in another study [3 [Recently, Zhang et al. reported that half of the 140 COVID-19 patients admitted to the hospital had eosinopenia  was observed in 75% of COVID-19 patients and more frequently  than in control patients [Besides the association of eosinopenia with the presence of COVID-19 or the severity of the disease, one relevant question for the management of COVID-19 is the triage of patients, which has been particularly challenging because of the large number of suspected patients admitted to the hospital and the long waiting time for rRT-PCR results and chest CT-scan availability. Therefore, it is necessary to develop more effective and accessible methods to optimize the triage process of these COVID-19 patients. In one study, the AUROC of eosinophils on admission to predict positive rRT-PCR was 0.77 [95%CI (0.641–0.886); es ratio ; also, tly i.e., % than inVery few diagnostic scores are available to rapidly identify patients with a high risk of positive rRT-PCR testing during COVID-19 pandemic. In one study, history of close contact, fever, neutrophil-to-lymphocyte ratio, high temperature and gender were included into a “COVID-19 early warning score”, which had a AUROC to diagnose a positive rRT-PCR of 0.96 ; howeverThis study has several limitations. First, this is a retrospective, single-centre study; as such, the results may suffer from selection biases. Moreover, we did not calculate the sample size to test the diagnostic accuracy of eosinopenia to predict a positive rRT-PCR test as, at the moment the study was initiated, the data on eosinopenia in COVID-19 were very limited and the prevalence of the positive rRT-PCR test in patients presenting in our institution was unpredictable. In particular, for a prevalence estimated around 50% of positive rRT-PCR testing, a minimum sample size of 365 patients would have been required to achieved a power of 80% in order to detect a change in the sensitivity from 0.80 to 0.90 and a change in the specificity from 0.6 to 0.7, with a significance level of 0.05. Future and multicentric studies are warranted to confirm our results using larger cohorts of patients. Second, some relevant laboratory variables might have been missed. Third, the sample size was not calculated and external validation not provided. Fourth, rRT-PCR could yield false negative results, mainly due to an insufficient viral load below the detection limit or inadequately collected nasopharyngeal swabs. Fifth, the clinical score was used locally and has not been validated in larger cohorts. Finally, the “pre-test” prevalence of COVID-19 in the analysed cohort could be a significant confounder to extrapolate the validity of our findings in clinical practice. Giving the high prevalence of COVID-19 among patients , it is difficult to conclude that eosinopenia is always related to a positive RT-PCR test for SARS-Cov-2. Indeed, these data should be re-evaluated during a phase of lower COVID-19 prevalence, where other inflammatory diseases could also be associated to initial eosinopenia and contribute as false positive. Moreover, we evaluated only hospitalized patients and the conclusions of our study cannot be applied to mild COVID-19 forms. In this study, eosinopenia was frequently observed in COVID-19 patients and could be helpful to identify patients with positive rRT-PCR testing or suspected chest CT-scan findings. This easily accessible parameter should be considered in the triage process to speed up the diagnosis and treatment of COVID-19 patients.


Polypoidal choroidal vasculopathy (PCV) is a type of age-related macular degeneration that can cause permanent vision loss. The purpose of this paper was to report the one-year outcomes of fixed-dosing aflibercept therapy for the treatment of PCV.This was a prospective, single-arm, interventional case series study of 25 PCV patients; 12 pre-treated and 13 treatment-naïve patients. The patients were treated and monitored for 12 months. Each patient was administered with an aflibercept (2.0 mg) injection every month for the first 3 months (the loading phase), and thereafter, once every 2 months. At every follow-up visit, best-corrected visual acuity (BCVA) test, fundus examination, and optical coherence tomography for measuring the central subfield macular thickness (CSMT) were performed. Fluorescein and indocyanine green angiography were conducted at baseline and at 4 and 12 months.p=0.001), and the CSMT significantly decreased from 406.92 um at baseline to 276.12 um (p< 0.001). Additionally, ten patients (40%) showed complete polyp regression. The treatment-naïve patients showed a statistically significant improvement in BCVA from 66.58 letters at baseline to 76.36 letters at 12 months, and a significant decrease in CSMT, from 462 to 243 um. In the pre-treated group, there was no change in BCVA (64.46 letters), and the decrease in CSMT from 356.08 to 303.69 um was not statistically significant.After 12 months of aflibercept therapy, the mean BCVA of the patients significantly improved from 65.48 letters at baseline to 69.91 letters , Republic of Korea. Identifer: KCT0005798, Registered: Jan 20, 2021. Retrospectively registered, URL:  Polypoidal choroidal vasculopathy (PCV) is a subtype of age-related macular degeneration (AMD). It is characterized by hemorrhage, serous exudates, and scar formation, all of which can lead to permanent vision loss . The incLimited studies have been conducted on the treatment regimen and natural progression of PCV compared with classic AMD . StudiesReported herein are the anatomical and functional outcomes of treating PCV patients with fixed dosing aflibercept therapy for 1 year. The data provided in this paper will be helpful in establishing a new PCV treatment protocol.This was a prospective, single-arm, single-center, interventional case series study conducted with the support of Bayer Korea, the manufacturer of aflibercept. This study was approved by the Nune Eye Hospital Institutional Review Board/Ethics Committee((IRB no: N-1312-001-023) and it complied with the tenets of the Declaration of Helsinki. All participants provided written informed consent.The study was conducted from June 2014 to March 2017. Initially, only treatment-naïve patients diagnosed with PCV for the first time were targeted. However, the inclusion criteria were revised to include pre-treated patients due to the limited number of treatment-naïve patients who could be enrolled in the study. Thirty patients were then included in the study and 25 patients completed a one-year follow-up. The inclusion and exclusion criteria are shown in Fig. Each patient received aflibercept (2.0 mg) injections (fixed dosing) in an aseptic operation room. For the loading phase, each patient was administered with an aflibercept injection once a month for 3 months; subsequently, maintenance injections were administered every 2 months. For 12 months, seven injections were administered at baseline and at the one-, two-, four-, six-, eight-, and ten-month follow-up visits. The treatment outcomes were finally evaluated at 12 months.At every follow-up visit after the initial baseline visit, best-corrected visual acuity (BCVA) was measured using the Early Treatment Diabetic Retinopathy Study (ETDRS) chart; intraocular pressure measurement, slit lamp examination, fundus photography, and optical coherence tomography  were performed as well. At baseline and at four and 12 months, fluorescein and indocyanine green angiography (ICGA)  were performed.The primary outcome measures assessed include the following: mean change in BCVA from baseline to 12 months, proportion of patients who had complete intraretinal/subretinal fluid resolution after 12 months, and proportion of patients who had complete polypoidal lesion disappearance after 12 months based on ICGA results.In evaluating polyp regression, complete disappearance of polyp was defined as complete regression, the decrease in the number of polyps was defined as partial regression, and no change in the number of polyps was defined as persistent polyp.p < 0.05.One researcher interpreted all the study images. For statistical analysis, the paired t-test, t-test, chi-squared test, and linear-by-linear association test were performed using the SPSS statistical software . Statistical significance was set at Altogether, 25 patients were enrolled in this study: 13 pre-treated patients and 12 treatment-naïve patients. For the 12 patients in the pre-treated group, the mean disease duration was 50.53±32.17 (5–96) months and the mean frequency of anti-VEGF injections was 18.23±15.41 (3–45) times; seven patients had a history of PDT, six of whom received PDT once and one, twice.p-value=0.045) and a higher number of polyps were observed . For the patients in the pre-treated group, most of their lesions were located outside the fovea , whereas for the patients in the treatment-naïve group, most of the lesions were located underneath the fovea    at the four-month follow-up, and in ten patients (40%) at 12-month follow-up Fig. .Fig. 3FAt 12 months, three patients (12%) showed a visual acuity improvement of 15 letters or more, and the number of patients with more than 20/40 visual acuity increased from 13 patients at baseline to 18 patients at 12 months , and a decrease of 218.45 um in central macular thickness, from 462±160 um at baseline to 243.55±162 um at 12 months . The pre-treated group also showed a decrease in central macular thickness, from 356.08±145 um at baseline to 303.69±139 um at 12 months; however, the change was not statistically significant . There was no change in the mean baseline visual acuity of the pre-treated group (64.46±16.52 letters) at 12 months. There was no significant difference in the complete polyp regression rates and complete subretinal fluid resolution rates of both the pre-treated and the treatment-naïve groups . The outcome of the EVEREST II study became the rationale for considering the combination with PDT as the first-line therapy for PCV . HoweverIn the present study, the treatment-naïve group had a 9.78-letter visual acuity improvement and a 41.6% complete polypoidal lesion regression rate. The visual acuity improvement was similar to what was recorded in the PLANET and VAULT studies, whereas the complete polypoidal lesion regression rate was similar to that of the PLANET study but lower than that of the VAULT study. In fact, the complete polypoidal lesion regression rates that has been published for aflibercept monotherapy for PCV is extremely variable, and has been reported to be up to 75% . AlthougThe pre-treated group in the present study did not show any statistically significant positive outcomes in terms of visual acuity improvement or decrease in retinal thickness decrease compared to the treatment-naïve group. In previous papers, improvement in visual acuity and decreased retinal thickness were recorded after the use of aflibercept for treating patients who did not respond to conventional therapies such as ranibizumab , 16; howThe present study had some limitations. Firstly, a control group was not included for comparison, and the study population was small. Secondly, PDT was not included as a treatment option. However, despite the limitations, the present study is significant in that it is a prospective study that highlights the usefulness of aflibercept monotherapy for PCV.In conclusion, the one-year fixed-dosing aflibercept therapy is a useful treatment with favorable anatomical and functional outcomes for treatment-naïve PCV patients. It also has favorable anatomical outcomes for pre-treated PCV patients. Larger studies with a longer follow-up period and a PDT group as a control group for comparison need to be conducted in the future to help formulate adequate PCV treatment guidelines.


TM system, and the data were analysed using the STATA statistical package. CTC number was presented as the mean values, and comparisons were made using the Student t-test. There was a trend toward increased CTC presence in the MSI-high (H) CRC group, but this was not statistically significant. In addition, a Poisson regression was performed adjusting for stage (I-IV). This demonstrated no significant difference between the two MSI groups for pre-operative time point t1. However, time points t2, t3, and t4 were associated with increased CTC presence for MSI-H CRCs. In conclusion, there was a trend toward increased CTC release pre-, intra-, and post-operatively in MSI-H CRCs, but this was only statistically significant intra-operatively. When adjusting for stage, MSI-H was associated with an increase in CTC numbers intra-operatively and post-operatively, but not pre-operatively.Microsatellite instability (MSI) in colorectal cancer (CRC) is a marker of immunogenicity and is associated with an increased abundance of tumour infiltrating lymphocytes (TILs). In this subgroup of colorectal cancer, it is unknown if these characteristics translate into a measurable difference in circulating tumour cell (CTC) release into peripheral circulation. This is the first study to compare MSI status with the prevalence of circulating CTCs in the peri-operative colorectal surgery setting. For this purpose, 20 patients who underwent CRC surgery with curative intent were enrolled in the study, and peripheral venous blood was collected at pre- (t1), intra- (t2), immediately post-operative (t3), and 14–16 h post-operative (t4) time points. Of these, one patient was excluded due to insufficient blood sample. CTCs were isolated from 19 patients using the Isoflux Biomarkers in colorectal cancer (CRC) have had limited success in clinical application to date, but microsatellite instability (MSI) status is emerging as a biomarker of clinical relevance. It is known that CRC exhibiting high level MSI (MSI-H) is associated with increased tumour infiltrating lymphocytes (TILs) and is a marker of immunogenicity ,2,3. MSIMicrosatellites are short tracts of repetitive sequence  found disseminated throughout the genome. Due to the repetitive nature of microsatellites, these regions are prone to change (instability) during replication. In MSI-H CRC, the resultant microsatellite alterations result in frameshifts that truncate proteins and may lead to inactivation of affected coding regions. Usually, microsatellite alterations are sensed by mismatch repair (MMR) genes that act like spellcheckers or DNA damage sensors, which detect mutations and signal for repair or apoptosis. When there is a loss of DNA damage sensors, either through genetic or epigenetic inactivation of MMR genes, this leads to loss of appropriate signalling and an accumulation of genetic mutations. In clinical practice, MSI-H occurs in 10–15% of colorectal cancers and is defined by IHC staining demonstrating MMR deficiency (MMRD).The serrated neoplastic pathway is one of the two sporadic pathways that result in MSI-H cancers, the other classical pathway being the adenoma-carcinoma pathway involving chromosomal instability (CIN) and that results in microsatellite stable (MSS) cancers. Interestingly, it appears that MSI-H colorectal cancers are less likely to progress to stage IV disease compared to their MSS counterpart ,5. HowevCTCs were first identified by Dr. Thomas Ashworth in 1869 .With the call for universal MSI testing in CRC, it is important to understand the immunobiology of MSI to understand its clinical implications and its role in guiding prognosis and adjuvant therapy. It is known that MSI is associated with TILs . HoweverTwenty patients undergoing elective laparoscopic or open colorectal surgery at either Liverpool or Westmead Hospitals were enrolled in the study approved by the South Western Sydney Local Health District Ethics (Ref: HREC/13/LPOOL/158). All patients gave informed written consent for blood collection and CTC analysis. Peripheral venous blood was collected at four time points: (t1) pre-operative blood collection in the anaesthetic bay of operating theatres; (t2) intra-operatively after mobilisation of bowel was completed; (t3) at time of completion of surgery; and (t4) fourteen-sixteen hours post-operatively. One patient had insufficient blood volume collected and was excluded from analysis.TM instrument . Peripheral venous blood was collected into 9 mL anti-coagulant K2EDTA tubes (Vacuette 455036) and processed within 24 h of collection in accordance with the Isoflux protocol using the CTC enrichment (910-0091) and enumeration (910-0093) kits supplied by the manufacturer. Briefly, immuno-magnetic EpCAM linked beads was used to capture CTCs, and after processing through the Isoflux instrument, CTCs were identified by immune staining using anti-cytokeratin , Hoechst 33342 dye, and anti-CD45. After transferring each sample to 24 well SensoPlatesTM  and applying coverslips, each sample was scanned and visualized using a 10× objective on a fluorescence Olympus IX71 inverted microscope. Putative CTCs were defined as CK+, DAPI+, and CD45−, nucleated and morphologically intact cells.Quantification of CTCs was performed using the IsoFluxCarcinoembryonic antigen (CEA), tumour infiltrating lymphocytes (TILs), microsatellite instability (MSI), and BRAF status were recorded. TILs were reported by the pathologist as present when there were more than 5 intraepithelial lymphocytes/100 epithelial cells (assessed on minimum three high power (×400) fields) . MSI andData on patient demographics, histopathological features of the tumour, and CTC count at four time periods were collected. Certified pathologists examined the tissue biopsy specimens post-operatively and provided the histopathology diagnosis. All patients had follow-up at one year with disease-free survival (DFS) being the main outcome.t-test was used to compare between groups, and a Poisson regression was used to adjust for stage. The Student t-test was used instead of the Wilcoxon–Mann–Whitney U-test and the Kruskal–Wallis tests as these non-parametric tests are better used to compare medians, whereas the Student t-test provides a better assessment of means.Analysis was performed on STATA . The Student TM system. Of the nineteen patients who had CTCs enumerated, two patients had high-grade dysplasia without malignancy (these were 30 × 33 × 23 mm and 57 × 50 × 55 mm villous adenomas). Of the remaining 17 patients, 3 (17.6%) were stage I, 6 (35.3%) stage II, 7 (41.2%) stage III, and 1 (5.9%) stage IV. Nine (52.9%) were right-sided , ascending colon (n = 4), and transverse colon (n = 1)), the rest (41.2%) were left-sided (rectum (n = 4), rectosigmoid (n = 1), sigmoid (n = 3)). The histopathology of all seventeen patients was adenocarcinoma. Further, two of the four patients with rectal cancer had neoadjuvant chemoradiotherapy. Patient demographic, clinical, and surgical characteristics are summarized in In total, 80 samples from 20 patients with colorectal cancer who underwent elective open or laparoscopic colorectal surgery with curative intent were recruited for this study. However, four samples from one patient had insufficient blood volume collected, and this patient was excluded from analysis. CTC isolation and enrichment were performed using the IsoFluxt-test. CTCs were enumerated for 19 patients: microsatellite stable (MSS) (n = 15); microsatellite unstable (MSI-H) (n = 4), respectively. A Student t-test was used to test the difference in CTC number between MSS and MSI-H CRCs, respectively, at the four different time points: t1 ; t2 ; t3 ; and t4 . It was apparent that at each of these time points, there was no significant difference between MSS and MSI-H patient groups.First, we looked at the number of CTCs enumerated for all 19 patients (p = 0.6191); t2 ; t3 ; t4 . In addition, a Poisson regression was performed adjusting for stage (I-IV). This demonstrated no significant difference between the two MSI groups for t1. However, t2, t3, and t4 were all associated with an increase in CTC number for MSI-H CRCs.Excluding the two patients with villous adenoma and high-grade dysplasia, there was a trend towards higher CTC number for MSI-H CRC, but this was not statistically significant between the two groups: t1 ; t2 ; t3 ; t4 ; p = 0.6027); t2 ; t3 ; t4 .For this analysis, there were no MSI-H patients in the stage I and IV groups; however, there were two MSI-H (caecum and ascending colon) patients in the stage II group; whereas there were four stage II MSS CRC  patients. For the stage II patients, there was a significant spike at the t2 timepoint for the MSI-H group (There was only one patient with stage IV colon cancer. This patient had a right-sided cancer that was MSS and had high CTCs that were persistently elevated with 13 CTCs detected at the pre-operative time point (t1), which increased to 189 during surgery and then remained high post-operatively with 83 and 65 CTCs detected, respectively .p = 0.6375); t2 ; t3 ; t4 ; There were no left-sided MSI-H CRCs in this study, so it was not possible to compare between MSI-H and MSS for left-sided colon and rectal cancer. However, there were four MSI-H and five MSS right-sided colon cancers. One MSS was excluded from analysis as it was a stage IV CRC. There was no statistically significant difference in CTC number for right-sided colon cancer by MSI status, but there was a trend for increased CTC number at t2, t3, and t4 time points  vs. 12.29 (8.88–15.71); t2: (20.99 (18.55–23.43) vs. 46.01 (38.18–53.83); t3: 7.72 (6.28–9.16) vs. 20.69 (13.88–27.49); t4: 8.03 (6.51–9.54) vs. 21.91 (16.62–27.18). In addition, a Poisson regression model was run for stage of CRC, and a post-estimation marginal fundamental analysis was performed. This showed a significant difference in CTC number at the t1, t2, t3, and t4 time points between stages independent of MSI status . PreoperWhen comparing the median CTC number between MSS and MSI-H CRCs, there was again a trend toward increased CTC number with MSI-H CRC, but this was only statistically significant at the t2 time point when comparing stage I-III MSS and MSI-H CRC.There is an abundance of clinical data reporting on the association between MSI status and prognosis in CRC showing that MSI-H may be associated with better prognosis ,4. The eOur study confirmed that CTC measurements correlated with dissemination and stage of disease. There was a statistically significant difference in CTC number with stage (I-IV) and across all time points . This demonstrated no significant difference between the two MSI groups for the t1 time point. However, the t2, t3, and t4 time points were all associated with increased CTC number for MSI-H CRCs.While most of the analyses performed were not statistically significant, overall, there was a trend towards increased CTC number at all time points  in the MSI-H group. When analysing stage II CRC and stage I-III CRC, there was a statistically significant increase in the CTC number for the t2 time point: 3.5 vs. 52, The literature on CTCs has shown that a measurement of more than three CTCs per 7.5 mL peripheral blood may be associated with poor survival, although some studies suggest 1–2 CTCs per 7.5 mL may also be associated with a worse outcome . FurtherWhat about the protective effect of MSI? The immunogenicity of MSI is believed to be associated with the presence of TILs. Collinearity between MSI-H status and TILs has been shown in clinicopathological data, including the data from our own cohort study , and hasHowever, this study showed that the immuno-biology of MSI-H CRCs is more complex than this. As is well established in the literature, MSI-H CRCs are also associated with poor differentiation ,37, are Notably, this pilot study showed results that warrant further investigations. The increased CTC positivity seen in MSI-H CRCs is a significant point of difference from the currently understood immune-biological mechanisms associated with MSI-H CRCs. In practice, this means that the benchmark cut-off points for CTC enumeration may be influenced by tumour characteristic, and future clinical applications of CTC in CRC management may need to take this into consideration. Another consideration is that the improved survival associated with immunogenic MSI-H CRCs may not be as profound as once believed, and the increased peri-operative release of CTCs in MSI-H CRCs may be revisited with future studies with a larger patient cohort. This could prove a useful follow up of the suggestions made by the European Society of Medical Oncology (ESMO) guidelines that patients with stage II colon cancer with MSI are at very low risk of recurrence and unlikely to benefit from chemotherapy.n = 20) was low and a limitation for insightful statistical analysis. There was significant heterogeneity within the study with two patients receiving neoadjuvant chemoradiotherapy, no direct comparisons available for stage I and IV (no MSI-H CRC in these subgroups), and the inclusion of right colon, left colon, and rectal cancers. The incidence of MSI in CRC is 10–15%. In our cohort, there were only four MSI-H CRCs, of which two were stage II and two were stage III, and the majority of patients were MSS CRCs, being a limitation of our data. From a tumour biology perspective, the main implication of our findings is that immunogenic MSI-H CRCs did not protect against release of CTCs, and the protective effect against metastatic disease was not by reducing CTCs. Further, the main clinical implication of this study involves the utility of CTCs for monitoring and surveillance with potentially differential baseline levels of CTCs associated with the two subtypes of CRC.In this study, the overall patient number (There was a trend toward increased CTC release pre-, intra-, and post-operatively in MSI-H CRCs, but this was only statistically significant intra-operatively. When adjusting for stage, MSI-H was associated with an increase in CTC number intra-operatively and post-operatively, but not pre-operatively. This dataset was limited, and further studies are required. Finally, these data suggested that immunogenic MSI-H CRCs did not suppress CTC release and that different reference ranges may be required for CTC enumeration of MSI-H and MSS CRC.